The double life of a chemotherapy drug: Immunomodulatory functions of gemcitabine in cancer.

Although the development of immunotherapies has been revolutionary in the treatment of several cancers, many cancer types remain unresponsive to immune-based treatment and are largely managed by chemotherapy drugs. However, chemotherapeutics are not infallible and are frequently rendered ineffective as resistance develops from prolonged exposure. Recent investigations have indicated that some chemotherapy drugs have additional functions beyond their normative cytotoxic capacity and are in fact immune-modifying agents. Of the pharmaceuticals with identified immune-editing properties, gemcitabine is well-studied and of interest to clinicians and scientists alike. Gemcitabine is a chemotherapy drug approved for the treatment of multiple cancers, including breast, lung, pancreatic, and ovarian. Because of its broad applications, relatively low toxicity profile, and history as a favorable combinatory partner, there is promise in the recharacterization of gemcitabine in the context of the immune system. Such efforts may allow the identification of suitable immunotherapeutic combinations, wherein gemcitabine can be used as a priming agent to improve immunotherapy efficacy in traditionally insensitive cancers. This review looks to highlight documented immunomodulatory abilities of one of the most well-known chemotherapy agents, gemcitabine, relating to its influence on cells and proteins of the immune system.

Gemcitabine Modulates HLA-I Regulation to Improve Tumor Antigen Presentation by Pancreatic Cancer Cells.

Pancreatic cancer is a lethal disease, harboring a five-year overall survival rate of only 13%. Current treatment approaches thus require modulation, with attention shifting towards liberating the stalled efficacy of immunotherapies. Select chemotherapy drugs which possess inherent immune-modifying behaviors could revitalize immune activity against pancreatic tumors and potentiate immunotherapeutic success. In this study, we characterized the influence of gemcitabine, a chemotherapy drug approved for the treatment of pancreatic cancer, on tumor antigen presentation by human leukocyte antigen class I (HLA-I). Gemcitabine increased pancreatic cancer cells' HLA-I mRNA transcripts, total protein, surface expression, and surface stability. Temperature-dependent assay results indicated that the increased HLA-I stability may be due to reduced binding of low affinity peptides. Mass spectrometry analysis confirmed changes in the HLA-I-presented peptide pool post-treatment, and computational predictions suggested improved affinity and immunogenicity of peptides displayed solely by gemcitabine-treated cells. Most of the gemcitabine-exclusive peptides were derived from unique source proteins, with a notable overrepresentation of translation-related proteins. Gemcitabine also increased expression of select immunoproteasome subunits, providing a plausible mechanism for its modulation of the HLA-I-bound peptidome. Our work supports continued investigation of immunotherapies, including peptide-based vaccines, to be used with gemcitabine as new combination treatment modalities for pancreatic cancer.

Communicating Native American Cancer Efforts and Resources in the Great Plains.

American Indians' cancer disparities vary by region, signifying the importance of understanding community-specific levels of readiness to address cancer. Twenty-four key respondents from an urban American Indian community in the Great Plains, community leaders (n = 8), educators (n = 8), and students (n = 8), were interviewed following the Community Readiness Model. Using framework analysis, we qualitatively examined data related to efforts and resources in interview transcripts. One overarching emergent theme and three subthemes were identified. We found that communication of efforts and resources is critical to increasing community readiness to address cancer and is most successful when the community (1) prioritizes cancer as an issue in the community, (2) cultivates trusting relationships among stakeholders, and (3) practices cultural responsiveness in efforts. These findings emphasize the need for better communication of cancer efforts and resources and can inform strategic efforts to promote cancer prevention, treatment, and research among American Indians/Alaska Natives in the Great Plains.

The histone deacetylase inhibitor M344 as a multifaceted therapy for pancreatic cancer.

The histone deacetylase (HDAC) inhibitor vorinostat, used with gemcitabine and other therapies, has been effective in treatment of experimental models of pancreatic cancer. In this study, we demonstrated that M344, an HDAC inhibitor, is efficacious against pancreatic cancer in vitro and in vivo, alone or with gemcitabine. By 24 hours post-treatment, M344 augments the population of pancreatic cancer cells in G1, and at a later time point (48 hours) it increases apoptosis. M344 inhibits histone H3 deacetylation and slows pancreatic cancer cell proliferation better than vorinostat, and it does not decrease the viability of a non-malignant cell line more than vorinostat. M344 also elevates pancreatic cancer cell major histocompatibility complex (MHC) class I molecule expression, potentially increasing the susceptibility of pancreatic cancer cells to T cell lysis. Taken together, our findings support further investigation of M344 as a pancreatic cancer treatment.

Immune checkpoint blockade in pancreatic cancer: Trudging through the immune desert.

Pancreatic cancer (PC) has exceptionally high mortality due to ineffective treatment strategies. Immunotherapy, which mobilizes the immune system to fight against cancer, has been proven successful in multiple cancers; however, its application in PC has met with limited success. In this review, we articulated that the pancreatic tumor microenvironment is immuno-suppressive with extensive infiltration by M2-macrophages and myeloid-derived suppressive cells but low numbers of cytotoxic T-cells. In addition, low mutational load and poor antigen processing, presentation, and recognition contribute to the limited response to immunotherapy in PC. Immune checkpoints, the critical targets for immunotherapy, have high expression in PC and stromal cells, regulated by tumor microenvironmental milieu (cytokine and metabolites) and cell-intrinsic mechanisms (epigenetic regulation, oncogenic signaling, and post-translational modifications). Combining immunotherapy with modulators of the tumor microenvironment may facilitate the development of novel therapeutic regimens to manage PC.

Major histocompatibility complex class I molecule expression by pancreatic cancer cells is regulated by activation and inhibition of the epidermal growth factor receptor.

Pancreatic cancer is one of the deadliest neoplasms, with a dismal 5-year survival rate of only 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. Downregulation of major histocompatibility complex (MHC) class I cell-surface expression can aid in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. Earlier studies suggested that epidermal growth factor receptor (EGFR) signaling can decrease MHC class I expression on certain cancer cell types. However, even though erlotinib (a tyrosine kinase inhibitor that targets EGFR) is an approved drug for advanced pancreatic cancer treatment, the impact of EGFR inhibition or stimulation on pancreatic cancer cell MHC class I surface expression has not previously been analyzed. In this current study, we discovered that EGFR affects MHC class I mRNA and protein expression by human pancreatic cancer cell lines. We demonstrated that cell-surface MHC class I expression is downregulated upon EGFR activation, and the MHC class I level at the surface is elevated following EGFR inhibition. Furthermore, we found that EGFR associates with MHC class I molecules. By defining a role in pancreatic cancer cells for activated EGFR in reducing MHC class I expression and by revealing that EGFR inhibitors can boost MHC class I expression, our work supports further investigation of combined usage of EGFR inhibitors with immunotherapies against pancreatic cancer.

Nanocarriers for pancreatic cancer imaging, treatments, and immunotherapies.

Pancreatic tumors are highly desmoplastic and immunosuppressive. Delivery and distribution of drugs within pancreatic tumors are compromised due to intrinsic physical and biochemical stresses that lead to increased interstitial fluid pressure, vascular compression, and hypoxia. Immunotherapy-based approaches, including therapeutic vaccines, immune checkpoint inhibition, CAR-T cell therapy, and adoptive T cell therapies, are challenged by an immunosuppressive tumor microenvironment. Together, extensive fibrosis and immunosuppression present major challenges to developing treatments for pancreatic cancer. In this context, nanoparticles have been extensively studied as delivery platforms and adjuvants for cancer and other disease therapies. Recent advances in nanotechnology have led to the development of multiple nanocarrier-based formulations that not only improve drug delivery but also enhance immunotherapy-based approaches for pancreatic cancer. This review discusses and critically analyzes the novel nanoscale strategies that have been used for drug delivery and immunomodulation to improve treatment efficacy, including newly emerging immunotherapy-based approaches. This review also presents important perspectives on future research directions that will guide the rational design of novel and robust nanoscale platforms to treat pancreatic tumors, particularly with respect to targeted therapies and immunotherapies. These insights will inform the next generation of clinical treatments to help patients manage this debilitating disease and enhance survival rates.

Collective Re-Storying of Mentee and Mentor Experiences in a Cancer Research Education Program for American Indian and Alaska Native Students.

The Youth Enjoy Science program at the University of Nebraska Medical Center has engaged American Indian/Alaska Native youth in mentored cancer research internships from 2017 to 2022. The primary purpose of this study was to examine mentor and mentee lived experiences of participation in Youth Enjoy Science research education internships and to provide insights that can inform mentorship practices in research education programs for American Indians/Alaska Natives. We conducted semi-structured interviews with current and former Youth Enjoy Science mentees (n=8) and mentors (n=8). Following a narrative inquiry research approach, we analyzed interview transcripts and collectively re-storied interview data. Participants described program characters, settings, problems, actions to address the problems identified, and resolutions that led to various recommendations for ways to raise contextual awareness between mentees and mentors.

Contribution of CXCR3-mediated signaling in the metastatic cascade of solid malignancies.

Metastasis is a significant cause of the mortality resulting from solid malignancies. The process of metastasis is complex and is regulated by numerous cancer cell-intrinsic and -extrinsic factors. CXCR3 is a chemokine receptor that is frequently expressed by cancer cells, endothelial cells and immune cells. CXCR3A signaling in cancer cells tends to promote the invasive and migratory phenotype of cancer cells. Indirectly, CXCR3 modulates the anti-tumor immune response resulting in variable effects that can permit or inhibit metastatic progression. Finally, the activity of CXCR3B in endothelial cells is generally angiostatic, which limits the access of cancer cells to key conduits to secondary sites. However, the interaction of these activities within a tumor and the presence of opposing CXCR3 splice variants clouds the picture of the role of CXCR3 in metastasis. Consequently, thorough analysis of the contributions of CXCR3 to cancer metastasis is necessary. This review is an in-depth examination of the involvement of CXCR3 in the metastatic process of solid malignancies.

Amyloid Precursor-like Protein 2 Expression Increases during Pancreatic Cancer Development and Shortens the Survival of a Spontaneous Mouse Model of Pancreatic Cancer.

In the United States, pancreatic cancer is a major cause of cancer-related deaths. Although substantial efforts have been made to understand pancreatic cancer biology and improve therapeutic efficacy, patients still face a bleak chance of survival. A greater understanding of pancreatic cancer development and the identification of novel treatment targets are desperately needed. Our analysis of gene expression data from patient samples showed an increase in amyloid precursor-like protein 2 (APLP2) expression within primary tumor epithelium relative to pancreatic intraepithelial neoplasia (PanIN) epithelial cells. Augmented expression of APLP2 in primary tumors compared to adjacent stroma was also observed. Genetically engineered mouse models of spontaneous pancreatic ductal adenocarcinoma were used to investigate APLP2's role in cancer development. We found that APLP2 expression intensifies significantly during pancreatic cancer initiation and progression in the LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model, as shown by immunohistochemistry analysis. In studies utilizing pancreas-specific heterozygous and homozygous knockout of APLP2 in the KPC mouse model background, we observed significantly prolonged survival and reduced metastatic progression of pancreatic cancer. These results demonstrate the importance of APLP2 in pancreatic cancer initiation and metastasis and indicate that APLP2 should be considered a potential therapeutic target for this disease.

The role of tumor heterogeneity in immune-tumor interactions.

The development of cancer stems from genetic instability and changes in genomic sequences, and hence, the heterogeneity exhibited by tumors is integral to the nature of cancer itself. Tumor heterogeneity can be further altered by factors that are not cancer cell intrinsic, i.e., by the microenvironment, including the patient's immune responses to tumors and administered therapies (immunotherapies, chemotherapies, and/or radiation therapies). The focus of this review is the impact of tumor heterogeneity on the interactions between immune cells and the tumor, taking into account that heterogeneity can exist at several levels. These levels include heterogeneity within an individual tumor, within an individual patient (particularly between the primary tumor and metastatic lesions), among the subtypes of a specific type of cancer, or within cancers that originate from different tissues. Because of the potential for immunity (either the natural immune system or via immunotherapeutics) to halt the progression of cancer, major clinical significance exists in understanding the impact of tumor heterogeneity on the associations between immune cells and tumor cells. Increased knowledge of why, whether, and how immune-tumor interactions occur provides the means to guide these interactions and improve outcomes for patients.

Tailoring Our Approach in Response to the SARS-CoV-2 Pandemic and Transcending Science Outreach Modalities for Native American Students in a Cancer Research Education Program.

In response to the SARS-CoV-2 pandemic, a cancer research education program at the University of Nebraska Medical Center designed for Native American middle school, high school and undergraduate students adapted activities to be delivered online. There are considerable challenges to adapting in-person science programming to online delivery that can impact overall effectiveness. These challenges are further exacerbated when the cognate student population also faces significant disparities in health, wealth, and educational outcomes. We encountered both disadvantages and advantages in transitioning programming to online virtual formats. Challenges faced in delivering our programming during the pandemic included varied online accessibility, peripheral stressors, and disconnection to places and people. Despite these challenges, we found several benefits in remote delivery, some of which have alleviated barriers to program participation for Native American students. Some successes achieved by transitioning to fully remote programming included increased program reach, sustainability, and cultural relevancy. In this overview of the implementation of four online programs at the middle school, high school, and undergraduate levels, we highlight the challenges and successes experienced. Through this program description, we aim to provide insight into potential strategies to improve program delivery designed for Native American students during the SARS-CoV-2 pandemic and beyond.

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4ß.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4ß-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4ß protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4ß-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4ß-loaded nanoparticles generated MUC4ß-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4ß vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Nanoformulation of CCL21 greatly increases its effectiveness as an immunotherapy for neuroblastoma.

Neuroblastoma is the most commonly diagnosed extracranial solid tumor in children. The patients with aggressive metastatic disease or refractory/relapsed neuroblastoma currently face a dismally low chance of survival. Thus, there is a great need for more effective therapies for this illness. In previous studies, we, as well as others, showed that the immune cell chemoattractant C-C motif chemokine ligand 21 (CCL21) is effective as an intratumoral therapy able to slow the growth of cancers. In this current study, we developed and tested an injectable, slow-release, uniform, and optimally loaded alginate nanoformulation of CCL21 as a means to provide prolonged intratumoral treatment. The alginate-nanoformulated CCL21, when injected intratumorally into mice bearing neuroblastoma lesions, significantly prolonged survival and decreased the tumor growth rate compared to CCL21 alone, empty nanoparticles, or buffer. Notably, we also observed complete tumor clearance and subsequent full protection against tumor rechallenge in 33% of nanoformulated CCL21-treated mice. Greater intratumoral presence of nanoformulated CCL21, compared to free CCL21, at days 1 and 2 after treatment ended was confirmed through fluorescent labeling and tracking. Nanoformulated CCL21-treated tumors exhibited a general pattern of prolonged increases in anti-tumor cytokines and relatively lower levels of pro-tumor cytokines in comparison to tumors treated with CCL21 alone or buffer only. Thus, this novel nanoformulation of CCL21 is an effective treatment for neuroblastoma, and may have potential for the delivery of CCL21 to other types of solid tumors in the future and as a slow-release delivery modality for other immunotherapies.

HLA-B influences integrin beta-1 expression and pancreatic cancer cell migration.

Human leukocyte antigen (HLA) class I molecules present antigenic peptides to cytotoxic T cells, causing lysis of malignant cells. Transplantation biology studies have implicated HLA class I molecules in cell migration, but there has been little evidence presented that they influence cancer cell migration, a contributing factor in metastasis. In this study, we examined the effect of HLA-B on pancreatic cancer cell migration. HLA-B siRNA transfection increased the migration of the S2-013 pancreatic cancer cells but, in contrast, reduced migration of the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Integrin molecules have previously been implicated in the upregulation of pancreatic cancer cell migration, and knockdown of HLA-B in S2-013 cells heightened the expression of integrin beta 1 (ITGB1), but in the PANC-1 and MIA PaCa-2 cells HLA-B knockdown diminished ITGB1 expression. A transmembrane sequence in an S2-013 HLA-B heavy chain matches a corresponding sequence in HLA-B in the BxPC-3 pancreatic cancer cell line, and knockdown of BxPC-3 HLA-B mimics the effect of S2-013 HLA-B knockdown on migration. In total, our findings indicate that HLA-B influences the expression of ITGB1 in pancreatic cancer cells, with concurrent distinctions in transmembrane sequences and effects on the migration of the cells.

Amphiphilic polyanhydride-based recombinant MUC4ß-nanovaccine activates dendritic cells.

Mucin 4 (MUC4) is a high molecular weight glycoprotein that is differentially overexpressed in pancreatic cancer (PC), functionally contributes to disease progression, and correlates with poor survival. Further, due to its aberrant glycosylation and extensive splicing, MUC4 is a potential target for cancer immunotherapy. Our previous studies have demonstrated the utility of amphiphilic polyanhydride nanoparticles as a useful platform for the development of protein-based prophylactic and therapeutic vaccines. In the present study, we encapsulated purified recombinant human MUC4-beta (MUC4ß) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4ß-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4ß-nanovaccine exhibited significant increase in the surface expressions of MHC I and MHC II and costimulatory molecules (CD80 and CD86), as well as, secretion of pro-inflammatory cytokines (IFN-γ, IL-6, and IL-12) as compared to cells exposed to MUC4ß alone or MUC4ß mixed with blank nanoparticles (MUC4ß+NP). Following immunization, as compared to the other formulations, MUC4ß-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4ß-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4ß-nanovaccine as a novel platform for PC immunotherapy.

Beta 2-microglobulin regulates amyloid precursor-like protein 2 expression and the migration of pancreatic cancer cells.

Beta 2-microglobulin (ß2m) is a component of the major histocompatibility complex (MHC) class I molecule, which presents tumor antigens to T lymphocytes to trigger cancer cell destruction. Notably, ß2m has been reported as persistently expressed, rather than down regulated, in some tumor types. For renal cell and oral squamous cell carcinomas, ß2m expression has been linked to increased migratory capabilities. The migratory ability of pancreatic cancer cells contributes to their metastatic tendencies and lethal nature. Therefore, in this study, we examined the impact of ß2m on pancreatic cancer cell migration. We found that ß2m protein is amply expressed in several human pancreatic cancer cell lines (S2-013, PANC-1, and MIA PaCa-2). Reducing ß2m expression by short interfering RNA (siRNA) transfection significantly slowed the migration of the PANC-1 and S2-013 cancer cell lines, but increased the migration of the MIA PaCa-2 cell line. The amyloid precursor-like protein 2 (APLP2) has been documented as contributing to pancreatic cancer cell migration, invasiveness, and metastasis. We have previously shown that ß2m/HLA class I/peptide complexes associate with APLP2 in S2-013 cells, and in this study we also detected their association in PANC-1 cells but not MIA PaCa-2 cells. In addition, siRNA down regulation of ß2m expression diminished the expression of APLP2 in S2-013 and PANC-1 but heightened the level of APLP2 in MIA PaCa-2 cells, consistent with our migration data and co-immunoprecipitation data. Thus, our findings indicate that ß2m regulates pancreatic cancer cell migration, and furthermore suggest that APLP2 is an intermediary in this process.

Cancer-associated mucins: role in immune modulation and metastasis.

Mucins (MUC) protect epithelial barriers from environmental insult to maintain homeostasis. However, their aberrant overexpression and glycosylation in various malignancies facilitate oncogenic events from inception to metastasis. Mucin-associated sialyl-Tn (sTn) antigens bind to various receptors present on the dendritic cells (DCs), macrophages, and natural killer (NK) cells, resulting in overall immunosuppression by either receptor masking or inhibition of cytolytic activity. MUC1-mediated interaction of tumor cells with innate immune cells hampers cross-presentation of processed antigens on MHC class I molecules. MUC1 and MUC16 bind siglecs and mask Toll-like receptors (TLRs), respectively, on DCs promoting an immature DC phenotype that in turn reduces T cell effector functions. Mucins, such as MUC1, MUC2, MUC4, and MUC16, interact with or form aggregates with neutrophils, macrophages, and platelets, conferring protection to cancer cells during hematological dissemination and facilitate their spread and colonization to the metastatic sites. On the contrary, poor glycosylation of MUC1 and MUC4 at the tandem repeat region (TR) generates cancer-specific immunodominant epitopes. The presence of MUC16 neo-antigen-specific T cell clones and anti-MUC1 antibodies in cancer patients suggests that mucins can serve as potential targets for developing cancer therapeutics. The present review summarizes the molecular events involved in mucin-mediated immunomodulation, and metastasis, as well as the utility of mucins as targets for cancer immunotherapy and radioimmunotherapy.

Cancer Biology and You: An Interactive Learning Event for Native American High School Students to Increase Their Understanding of Cancer Causes, Prevention, and Treatment, and to Foster an Interest in Cancer-Related Careers.

The National Institutes of Health and the National Science Foundation have made a compelling call to action not only to strengthen the pipeline of available STEM-trained talent, but in addition to foster students who are members of populations currently under-represented in science. Furthermore, the scientific community must not only increase the accessibility of STEM-related education, but also implement and test evidence-based practices. Presented here, we detail the proceedings of a hands-on, science-focused informal learning opportunity aimed at educating an underrepresented population in cancer biology. Fifteen undergraduate and graduate student volunteer instructors from the University of Nebraska at Omaha and the University of Nebraska Medical Center engaged with 89 high school students, mostly Native American, in an informal learning event called "Cancer Biology and You Day." Throughout the event, students completed two independent lessons focusing on breast cancer and skin cancer and demonstrated strong learning gains associated with the lessons as assessed by KWL charts. Exit surveys of the students indicated high levels of satisfaction with the event, and positive attitudes associated with considering a career in science/research were evident in survey responses. Overall, we report the event as a success and outline how similar experiences may be achieved.